The study of tissues for medical research, disease diagnosis, disease prognosis as well as the examination of tissues after in vivo or in vitro exposure to agents of interest requires the ability to examine the intact tissue in three dimensions at all depths. For example, it is useful to be able to examine an entire lobe of an animal's lung where the animal has been exposed to an inhaleable substance, such as insulation fiber, in order to determine the distribution, biopersistence and pulmonary toxicity of the substance in the lung tissue.
Whole mount imaging of organisms or tissues provides an immediate and three-dimensional view of the architecture and morphology of the organism or tissue. Current technology in confocal microscopy and deconvolution technology allow the visualization of different focal planes within a sample of interest. Using these techniques, a three dimensional reconstruction of a biological sample can be made. However, these technologies are limited to relatively thin sections of sample. Under normal imaging conditions, the depth of optical penetration into specimens is typically limited to 20-40 microns.
Three-dimensional information can also be gained by serial section analysis, but only following an indirect, time-consuming process. In addition, sectioning is subject to many preparation artifacts that can distort the morphology of the tissues under study and disrupt or distort the distribution of the agent of interest within the tissue.
The methods used to achieve controlled exposure of animals to substance of interest often introduce method specific artifacts, necessitating the use of more than one method of exposure in any given study. Current fiber imaging methodologies, such as ash analysis, limit important observations and interpretations to bulk fiber load and size class of fibers, and do not reveal fiber position or associations with relevant pulmonary structures, important to lung pathology. Furthermore, traditional methods, such as ash analysis, can destroy pertinent information, while other methods, such as serial sectioning can take a year or more to complete thorough analysis of a specimen.
For example, pulmonary toxicity of natural and man made fibers are assessed in animal models using two-exposure methods: aerosol and intratracheal instillation. While each has distinct advantages, under certain conditions, one method may be more appropriate than the other. When the precise dose of a material is required, intratracheal instillation may be the preferred method of administration. However, the possibility of uneven distribution induced by fiber agglomeration may cloud interpretation of results. Although many studies have addressed clearance of fibers in lungs, none have adequately addressed the problem of fiber agglomeration in airways resulting from the method of fiber administration to lungs.